JPH0147155B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing glutathione, and more particularly to a method for producing glutathione using a microorganism belonging to the genus Escherichia in which the control mechanism of the glutathione synthase system has been released. Glutathione is a tripeptide consisting of L-glutamic acid, L-cysteine and glucine.
It is a substance useful as a treatment for liver diseases, an antidote, etc. It is also a substance useful as a biochemical reagent. Conventionally, methods for producing glutathione using microorganisms include a method of extracting glutathione from yeast cells, and a method of contacting dry yeast with good membrane permeability with a substrate solution containing L-glutamic acid, L-cysteine, and glycine. Known methods include methods for producing glutathione, and methods for conjugating an ATP regeneration system when producing glutathione by contacting yeast or Escherichia coli cells with a substrate solution. However, when viewed as an industrial production method, these methods do not necessarily produce a satisfactory amount of glutathione. In view of this situation, the present inventors have conducted various studies to find a method for industrially advantageous production of glutathione using microorganisms, and have found that glutathione belongs to the genus Escherichia. We have discovered that microorganisms that have glutathione activity and have released the inhibition of γ-glutamylcysteine synthetase can serve as an excellent enzyme source for producing glutathione from L-glutamic acid, L-cysteine, and glycine. The invention was completed. That is, the present invention involves culturing the above-mentioned microorganisms, and using the thus obtained microorganisms or the processed product of the microorganisms as L-
This is a method for producing glutathione, which comprises contacting with a substrate solution containing glutamic acid, L-cysteine, and glycine and collecting the produced glutathione. The microorganism used in the present invention belongs to the genus Escherichia and has γ-glutamylcysteine synthase (E.
C.6.3.2.2., hereinafter referred to as GSH-) activity and glutathione synthase (EC6.2.3.2., hereinafter referred to as GSH-) activity, and has glutathione-induced GSH- Any microorganism that has been released from inhibition can be used, and a typical example of such a microorganism is GSH-
Suitable examples include Escherichia coli which has GSH-activity and GSH-activity and whose GSH-activity is released from inhibition by glutathione. The above bacterial strain can be obtained, for example, as follows. First, GSH-activity and a wild strain of E. coli with GSH-activity (e.g.
A cysteine-requiring strain and a methylglyoxal-resistant strain are obtained by inducing mutations in Escherichia coli B355). Mutations can be induced by conventional mutagenesis treatments, such as N-methyl-
This can be carried out by treatment with a mutagenic agent such as N'-nitro-N-nitrosoguanidine. To obtain a cysteine auxotroph, the resulting strain is subjected to mutagenesis in a minimal medium containing 2×10 ^-5 M cysteine (e.g., K ₂ HPO ₄ 0.7%, KH ₂ PO ₄ 0.3%,
It can be obtained by culturing in a medium containing 0.1% (NH ₄ ) ₂ SO ₄ and 0.5% glucose (hereinafter referred to as DM medium) and separating the resulting small colonies. On the other hand, a methylglyoxal-resistant strain can be obtained by culturing the strain obtained by the above mutagenesis treatment in a DM medium containing methylglyoxal, and separating the resulting large colonies by fishing. Next, the above-mentioned methylglyoxal-resistant strain is cultured in a minimal medium containing the cysteine-requiring strain obtained above, and colonies that do not form a halo around the colony are isolated to obtain a GSH-deficient strain (e.g., Escherichia coli C912). get. Next, mutations were induced in this GSH-deficient strain using the same treatment methods as described above, followed by culturing it in a DM medium containing 8-hydroxyquinoline, and the resulting colonies were isolated by fishing to induce GSH-deficient strains.
It is possible to obtain a strain that has GSH-activity and GSH-activity, and in which the inhibition of GSH- by glutathione is released. Examples of strains obtained in this way include, for example, Escherichia P. coli.
An example is RC912 (Feikoken Jokyo No. 47). The medium used for culturing the microorganism of the present invention obtained as described above includes a carbon source,
Either a synthetic medium or a natural medium can be used as long as it contains a suitable amount of nitrogen sources, inorganic substances, etc. As the carbon source, various hydrocarbons such as glucose, sucrose, fructose, starch, starch hydrolyzate, and molasses can be used, and the amount used is preferably about 0.5 to 5.0%. Examples of nitrogen sources include various inorganic and organic ammoniums such as ammonium sulfate, ammonium phosphate, ammonium carbonate, and ammonium acetate;
Alternatively, nitrogenous organic substances such as peptone, yeast extract, corn steep liquor, and casein hydrolyzate can be used, and the amount used is preferably about 0.5 to 2.0%. Further, as the inorganic substance, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, etc. can be used, and the amount used is preferably about 0.005 to 0.5%. The culture is preferably carried out under aerobic conditions such as shaking culture or aeration agitation culture. The suitable culture temperature is 25-37℃, and the culture period is usually 16-40℃.
About an hour is enough. After the cultivation is completed, the grown bacterial cells are collected, and the bacterial cells or a processed product of the bacterial cells are used as an enzyme source. Examples of processed products of bacterial cells include, for example, dried bacterial cells, cell-free extracts obtained by ultrasonication, enzymes purified from the extracts, etc., as well as bacterial cells or purified enzymes that are encased in polyacrylamide gels. Preferred examples include immobilized bacterial cells and immobilized enzymes obtained by immobilization by a method such as a carrageenan gel entrapment method or the like. Glutathione can be produced by bringing the enzyme source as described above into contact with a substrate solution containing L-glutamic acid, L-cysteine, and glycine to cause an enzymatic reaction. The concentration of L-glutamic acid in the substrate solution is preferably about 5 to 50 mM, the concentration of L-cysteine is about 5 to 50 mM, and the concentration of glycine is about 50 to 100 mM. This enzymatic reaction is preferably carried out at a pH of 7 to 8.5, and the reaction temperature is 20 to 50°C, especially 30°C.
~37°C is preferred. In addition, in order to carry out this enzymatic reaction efficiently, this enzymatic reaction is carried out using adenosine-5'-
It is preferable to conjugate it with a phosphoric acid (ATP) regeneration system. As the ATP regeneration system, reactions by acetokinase, glycolytic enzymes, carbamyl phosphate kinase, pyruvate kinase, etc. possessed by the microorganism of the present invention can be used. For example, when using the reaction by acetokinase, the substrate Magnesium ions are added to the solution as magnesium salts (e.g. magnesium sulfate, magnesium chloride).
20mM adenosine-5'-triphosphate (ATP)
It is preferable that about 5 to 10 mM of acetyl phosphate be present. Glutathione is thus produced and accumulated in the reaction solution, but the produced glutathione can be isolated and purified by a known method such as treatment with an ion exchange resin. For example, after adjusting the reaction completion solution to pH 3 with sulfuric acid, a cation exchange resin (e.g. Diaion PK-
228H ⁺ ) conducts. The adsorbed glutathione
Elute with 0.5M ammonium hydroxide, adjust the eluate to pH 4.5 with sulfuric acid, and then add anion exchange resin (e.g.
Duolite A2 CH ₃ COO ^- type) conducts. Elute the adsorbed glutathione with 0.5M sulfuric acid and add it to the eluate.
Glutathione can be obtained by adding 50% ethanol and isolating the precipitated crystals. Examples of the present invention will be shown below. Example 1 The strains shown in Table 1 below were mixed with 0.5% glucose, 0.3% potassium hydrogen phosphate, 0.7% potassium dihydrogen phosphate, and 0.01% magnesium sulfate heptahydrate.
%, ammonium sulfate 0.1% (PH
7.0) and cultured with shaking at 37°C for 16 hours.
After the culture was completed, the bacterial cells were collected by centrifugation, and a cell-free extract was prepared by sonication. Add 0.05ml of this cell-free extract to 25mM of L-glutamic acid,
Cysteine 25mM, glycine 50mM, magnesium chloride 10mM, acetyl phosphate 10mM and
The mixture was added to 9.95ml of 50mM Tris buffer containing 5mM of ATP, and enzymatic reaction was performed at 37°C for 1 hour. After the reaction is complete,
Quantify the amount of glutathione in the reaction solution, and
The amount of glutathione produced per mg was calculated. The results were as shown in Table 1 below. 【table】

Claims (1)

[Scope of Claims] 1. A microorganism belonging to the genus Escherichia that has γ-glutamylcysteine synthetase activity and glutathione synthetase activity and in which inhibition of γ-glutamylcysteine synthetase by glutathione has been released is cultured, A method for producing glutathione, which comprises contacting the bacterial cells or a processed product of the bacterial cells with a substrate solution containing L-glutamic acid, L-cysteine and glycine, and collecting the produced glutathione. 2. The production method according to claim 1, wherein the microorganism is Escherichia coli which has γ-glutamylcysteine synthetase activity and glutathione synthetase activity and in which inhibition of γ-glutamylcysteine synthetase by glutathione has been released. . 3. The production according to claim 1 or 2, in which bacterial cells or a processed product of the bacterial cells are brought into contact with a substrate solution in the presence of magnesium ions, adenosine-5'-triphosphate, and acetyl phosphate. Law.